Cryptosporidium parvum is an opportunistic intestinal protozoan parasite which infect AIDS patients and other immune-compromised individuals. For lack of effective drugs, cryptosporidiosis may become persistent, particularly in immunodefficient individuals. Genes controlling virulence phenotypes in this parasite are unknown, but their identification is needed to guide the development of drugs, to better understand pathogenesis, and to improve the annotation of the genome. The long-term objective of this revised application is to apply genetic methods to map genes controlling specific phenotypes. In contrast to linkage analysis used in the first funding period, we propose to apply Linkage Group Selection, a strategy which does not require the cloning of progeny lines. Instead, genetic loci controlling selectable traits are identified by crossing two phenotypically distinct C. parvum lines, exposing progeny populations to one or multiple rounds of selection, and quantitatively genotyping progeny populations in bulk. Based on well-defined phenotypic differences between the parental lines, we will select for early oocyst differentiation, oocyst resistance to bleach, and parasite infectivity to interferon-? knock-out mice. During selection, parasites carrying alleles conferring the phenotype selected against will be eliminated from the progeny. As a result of meiotic recombination the proportion of alleles from the sensitive parent will increase with distance from the locus under selection, giving rise to a [unreadable]selection valley[unreadable]. Such valleys will be identified by quantitatively genotyping a selected and a control progeny population with a panel of single nucleotide polymorphisms. This study will be performed in collaboration with Dr Paul Hunt, a malaria geneticist who has developed and successfully applied Linkage Group Selection to the study of malaria drug resistance. Specific Aims: 1. Cross phenotypically distinct isolates of Cryptosporidium parvum and use Linkage Group Selection to identify chromosomal regions controlling specific phenotypes. Populations of uncloned recombinant parasites derived from crosses between phenotypically distinct parental lines will be subjected to selective pressure and quantitatively genotyped using a set of 100 SNP markers. Regions under selection will be progressively resolved by iterative recombination and selection. 2. Identify genes within a selection valley controlling the phenotype under selection and identify the mutations responsible for the phenotype. A set of closely spaced SNPs located within the selection valley will be proportionally sequenced to identify genes and mutations under selection. Mutations will be mapped to the genome of C. hominis, C. muris and more distantly related apicomplexan to identify those most likely to affect protein function or transcriptional regulation.